Cloning and cDNA Synthesis of PE & PPE Gene of Mycobacterium Strain H37rv5

International Journal of Medical, Pharmacy and Drug Research (IJMPD)
[Vol-6, Issue-2, Mar-Apr, 2022]
ISSN: 2456-8015
https://dx.doi.org/10.22161/ijmpd.6.2.6
Peer-Reviewed Journal
Int. J. Med. Phar. Drug Re. 2022 42
Vol-6, Issue-2; Online Available at: https://www.aipublications.com/ijmpd/
Cloning and cDNA Synthesis of PE & PPE Gene of
Mycobacterium Strain H37rv5
Utkalendu Suvendusekhar Samantaray, BSc, MSc
1
; Rudra Prasad Khuntia2
1
Utkal Univeristy, Bhubaneswar, Odisha, India
Email: Utkalgenome@gmail.com
2
Siksha 'O' Anusandhan Deemed to be University, Odisha, India
Email; rudraprasad8018@gmail.com
Received: 01 Feb 2022; Received in revised form: 01 Mar 2022; Accepted: 09 Mar 2022; Available online: 20 Mar 2022
©2022 The Author(s). Published by AI Publications. This is an open access article under the CC BY license
(https://creativecommons.org/licenses/by/4.0/)
Abstract— Pathogenic, slow-growing Mycobacterium tuberculosis and other Mycobacterium tuberculosis
complex (MTBC) species include the pe/ppe genes. In the pathogen-host connection, these genes are crucial.
Despite the fact that the activities of most PE/PPE family proteins are unknown, mounting evidence shows
they play a role in M. tuberculosis infection. The role of PE/PPE proteins, which are thought to be involved
in the ESX system's action, is investigated. During complicated host-pathogen processes, we also discuss
how PE/ PPE proteins are involved in host-pathogen interactions, immune response regulation, and cell fate
determination. Finally, we discuss future PE/PPE protein research priorities as well as how present
information might be used to develop more accurate diagnostics and vaccines for worldwide TB control. The
pe and ppe genes, which are thought to be critical in TB pathogenesis, are only found in mycobacteria.
However, nothing is known about how these genes' expression is regulated. Understanding the regulatory
control of genes present only in mycobacteria, such as the pe and ppe gene families, might help researchers
figure out why the illness is so effective. A transposon mutagenesis method was employed to better
understand pe and ppe control. Rv1403c, a previously unknown transcriptional regulator, was discovered
as a consequence of this study.
Keywords— Cloning, Mycobacterium tuberculosis, H37Rv.
I. INTRODUCTION
Mycobacterium tuberculosis (M. tb) is a TB-causing
Mycobacteriaceae pathogenic bacteria. Due to the presence
of mycolic acid, which was first identified by Robert Koch
in 1882, M. tuberculosis has a distinctive waxy coating on
its cell surface. M. tuberculosis may look Gram-negative or
Gram-positive due to this coating, which makes the cells
resistant to Gram staining. Instead, M. tuberculosis is
detected using a microscope using acid-fast stains like
Ziehl–Neelsen or fluorescent stains like auramine. To live,
M. tuberculosis has an aerobic physiology that needs a lot
of oxygen. It is mostly a mammalian respiratory disease
affecting the lungs. The most common tuberculin tests
include the tuberculin skin test, acid-fast stain, culture, and
polymerase chain reaction. M. tuberculosis causes
tuberculosis, which is an airborne infection. One-third of the
world's population is thought to be latently infected with M.
tuberculosis, with three million people dying from the
illness each year. The growth of drug-resistant strains is
exacerbating the problem. Despite substantial research, the
pathophysiology, virulence, and durability of M.
tuberculosis infection remain unclear. A significant area in
TB pathogenesis is the discovery of M. tuberculosis
virulence factors that are related to human sickness.
Weakening mycobacteria strains might be utilized to
uncover genes that are critical for pathogenesis. The most
well-studied virulent laboratory strain of M. tuberculosis is
H37Rv. It is infectious, but it does not replicate in
macrophages, and it is similar to M. tuberculosis in that it
does not replicate in macrophages during latent infection.
The causes for the reduced pathogenicity are unclear at this
time. The genetic and behavioral differences between these
strains have been widely researched in the hopes of
discovering virulence factors. M. tuberculosis H37Rv has a

Samantaray et al./ Cloning and cDNA Synthesis of PE & PPE Gene of Mycobacterium Strain H37rv5
faster in vivo growth rate than M. tuberculosis H37Ra, and
the better intracellular survival gene and exported repetitive
protein genes help M. tuberculosis survive in macrophages.
Genomic alterations such as insertions, deletions, and single
nucleotide polymorphisms have been identified in both
virulent and attenuated Mycobacteria, in addition to
recognized virulence factors. Other studies looked at the
phenotypic consequences and changes in gene expression
independent of the genetic differences between H37Ra and
H37Rv. In M. tuberculosis H37Ra, several genes were
identified to be repressed. As a consequence, although
finding genes connected to M. tuberculosis virulence is
critical, the gene products at the protein level that are
responsible for virulence should also be prioritized. For
identifying whether genes are genuinely expressed,
proteomics characterization is a significant adjunct to
genomics. Thanks to enhanced label-free processes,
proteomic technologies have recently taken on a new
dimension. Proteins that are differentially expressed, such
as proteins that are up-regulated or down-regulated, as well
as proteins that are impacted by shaking culture conditions,
may be found in the BCG proteome. This is something that
no amount of genetic study will be able to uncover.
Furthermore, proteomics of M. tuberculosis H37Rv
revealed six open reading frames that had not been predicted
by genomics. Differences in protein composition between
attenuated and virulent M. tuberculosis strains may help
researchers design novel vaccines and therapies. M.
tuberculosis is an intracellular pathogen found in the host's
macrophages. When M. tuberculosis infects host cells, it
uses membrane and surface proteins to communicate with
them. These proteins are hypothesized to be involved in
intracellular proliferation and the bacterial response to
microbicidal actions in the host. M. tuberculosis possesses
a real outer membrane, which adds to the complexity of
bacterial-host interactions and gives important information
on anti-mycobacterial therapeutic sensitivity.
II. REVIEW OF LITERATURE
GENERAL FEATURES OF PP AND PPE GENES
It's vital to understand the features that determine a protein's
function before looking at it. Separate the protein of interest
into the conserved PE or PPE domains and the highly
variable C-terminal domains as a first step in investigating
PE/PPE proteins. Despite the fact that their function is
mostly unclear, molecular and biochemical investigations
have substantially enhanced our knowledge of the PE/PPE
domains. All PE proteins have a highly conserved N-
terminus, which consists of roughly 100 amino acids
organized in a helix-turn-helix sequence. The ten N-
terminal amino acids include the pro-glu residues that give
the protein its name, whereas the PE-C-terminus domain
contains the conserved YxxxD/E TypeVII secretion signal.
This secretion signal is necessary for the secretion of these
proteins and may have a role in TypeVII secretion systems'
substrate identification. PPE proteins share a lot of
characteristics. The pro-pro-glu motif may be found
towards the N-terminus of the PPE domain, which is
generally roughly 180 amino acids long. Because PE and
PPE proteins' amino acid sequences are not always similar,
this should not be used as the only criteria for identifying
PPE proteins. All PPE proteins contain the WxG motif
between the second and third alpha helix, but they do not
have the YxxxD/E secretion. PE and PPE proteins were
shown to be secreted as heterodimers in all studies with
sufficient biochemical characterisation. The three N-
terminal alpha-helices of the PPE protein form hydrophobic
contacts with the homologous PE protein. The conserved
WxG of the PPE-protein and the YxxxD/E of the PE protein
are near together in this heterodimeric structure, and these
sequences are expected to create a composite recognition
structure for TypeVII secretion. This PE/PPE complex
resembles Esx protein heterodimers, such as EsxA-EsxB
(ESAT-6/CFP10), or even Esx protein homodimers in
Staphylococcus aureus. PE/PPE proteins have a structure
that is very similar to the ESX-1 secretion-associated
proteins (esp) family's EspA/C and EspE/F heterodimers, as
well as EspB. The helix-bundle structure and composite
secretion signal are unique features of PE/PPE heterodimers
and other TypeVII-associated substrates, according to these
studies.
SUBCLASSIFICATION OF PE/PPE PROTEINS
The C-terminal domains of PE/PPE proteins contain the
bulk of the protein's variation and, potentially, the most
fascinating properties. By splitting PE/PPE proteins into
subgroups, these C-terminal domains may also assist
researchers build viable study hypotheses and give
functional or evolutionary insight. The polymorphic gc-rich
sequences (PGRS) are the most common kind of PE protein,
accounting for 65 of the 99 found in the M. tuberculosis
reference genome H37Rv. The PE PGRS subgroup is the
most challenging to analyze, with gccontents over 80% in
certain instances, hydrophobic glycine-rich repetitions, and
enormous sizes (up to 1400 amino acids). Based on C-
terminal sequences, the majority of the PE-proteins cannot
be clearly subgrouped into separate functional groups. The
PE-domain sequences, on the other hand, and their genetic
background provide further information. Gey van Pittius et
al. classified PE-proteins into five sublineages based on
their sequence phylogeny. Sublineage I and II relate to the
ancestral pe-genes encoded by ESX-1 (PE35) and ESX-3
(PE5), respectively. Sublineage III, which comprises the
ESX-5 substrate PE25 as well as the esx2-localized PE36,

is the most loosely defined sublineage. Sublineage IV refers
to a subset of secreted PE proteins from ESX-5, whose
genes are frequently bicistronic transcripts with PPE-SVP
genes. All PE PGRS proteins are found in Sublineage V, as
well as a variety of proteins with various C-terminal
domains, including putative hydrolases, lipases, and
cutinases, the most well-known of which is LipY. Phyre2
was used to identify a variety of additional PE proteins
(PE1, PE3, PE4, and PE16) as potential hydrolases (three
closest hits with 99 percent confidence: PDB 3AJA, 1QOZ,
and 3HC7).
The PF08237 domain, dubbed the 'PE/PPE domain' by
Adindla and Guruprasad, is a hydrolase-like fold. The
PFAM08237 domain has also been discovered in the PPE
proteins PPE28, PPE42, and PPE63, as well as non-TypeVII
substrate proteins, suggesting that these putative enzymatic
domains may have evolved into TypeVII substrates via
genetic recombination with the pe and ppe genes. PE2,
which also contains PFAM08237, is unlikely to be a true PE
protein since it lacks any of the PE, YxxxD/E, or helix-turn-
helix domains. In M. tuberculosis, LipY is a PE protein, but
in M. marinum, it is a PPE protein. According to the finding
of these ESX-5-secreted lipases, putative hydrolases, and
cutinases, TypeVII-secreted PE and PPE proteins may be
involved in cell envelope production and homeostasis, as
well as lipid-based nutrition absorption.
Within the PPE protein, there are even more subclasses. The
PPE-PPW proteins are the first well-identified subclass,
aside from the ESX-1-specific (PPE68) and ESX-2-specific
(PPE69) PPEs. Several proteins have a PxxPxxW amino
acid motif between 10 and 30 amino acids from the C-
terminus. M. tuberculosis H37Rv has ten PPW proteins,
despite the fact that PPE48 and PPE67 are truncated
proteins that may be detrimental. With 26 members, the
PPE-SVP proteins are the largest subgroup of PPE proteins,
named after the conserved SVP (Serine-Valine-Proline)
amino acid sequence present in their C-terminal domain.
PPE50 is most likely a truncated protein, and PPE9 is not a
true PPE-SVP because it only includes a PPE domain of 180
amino acids, although it was identified as such by
phylogenetic analysis. The size of the remaining 24 PPEs
varies from 350 to 468 amino acids.
Finally, PPE-MPTR proteins that include the Major
Polymorphic Tandem Repeat (MPTR) are the most recently
produced subclass of PPE proteins. The MPTR-repeat is
made up of NxGxGNxG motif repeats that vary in size and
form. While some PPE-MPTR proteins are small, others in
the family have released proteins that may be as large as
3716 amino acids.
SECRETIONS OF PP AND PPE GENES
To begin with, although all pe/ppe genes are often omitted
from bioinformatic databases, in the great majority of
situations, this is an unnecessarily strict approach. Even
short-read sequencing techniques can successfully map
practically all pe/ppe genes if only pe pgrs and ppe-mptr
genes/transcripts are excluded, thanks to paired-end
technology and longer read lengths. Understanding a
PE/subgroup PPE's may also be a great place to start when
guessing on the most probable route of secretion, and may
even indicate roles or redundancy. Individual proteins, even
within the groupings indicated below, may behave quite
differently from one another.
As a consequence, subgrouping may aid in the generation
of testable concepts, however these should always be
experimentally confirmed before drawing firm conclusions.
PPE4, which is genetically encoded inside the ESX-3
chromosomal region and is likely secreted through this
secretion route with its secretion partner PE5, is the best-
studied member of the PPE-PPW proteins. Mycobactin-
mediated iron absorption requires these proteins. In one
experiment, the PPE-PPW PPE20 and its cognate partner
PE15 were discovered to be ESX-3 substrates. PPE36 and
PPE37, two more PPW proteins, are known to have a role
in iron homeostasis through heme-iron acquisition
activities. These results, together with a sequence study of
the EspG-binding region, indicate that PPE-PPW proteins
use ESX-3 rather than ESX-5 for translocation.
Experimental investigations show that ESX-5 secretes all
PPE-SVP proteins. PPE-SVP proteins are often transcribed
as bicistronic transcripts including a sublineage IV PE-
protein and two Esx-proteins. These operonic clusters are
most likely the first pe/ppe genes transferred from the ESX
genetic locus when the ESX-5 system was introduced into
the slow-growing mycobacteria's most recent common
ancestor. PE/PPE proteins are encoded by these operons.
These operons generate PE/PPE proteins that are
exclusively secreted as PE/PPE heterodimers and are not
promiscuous with other secretion partners. The PE8-
PPE15-EsxI-EsxJ operon, which has been discovered to
increase protein secretion, is an outstanding example of
such an operon structure.
H37R5 STRAIN
Mycobacterium tuberculosis strain H37Rv is the most
studied TB strain in research facilities. In 1905, Dr. Edward
R. Baldwin became the first to isolate it. The strain was
discovered in a 19-year-old patient with chronic pulmonary
tuberculosis at the Trudeau Sanatorium in Saranac Lake,
New York. It was initially identified as strain H37, and it
was maintained alive at the Trudeau Sanatorium for many
years using serial culture transfer. It was observed that the
virulence of it in animal models varied according on the

substrate it was cultivated on throughout time. After that,
strains with different levels of virulence were produced on
purpose, with H37R being less virulent after growing in
acidic media and H37S being more virulent after growing
in alkaline media in guinea pigs (with R standing for
resistant to environment, and S for sensitive to
environment). The more virulent strain was subsequently
given the name H37Rv, with R designating rough
morphology and v denoting virulent. The strain was used in
a number of laboratory studies before finally becoming the
TB standard. Koch first identified Mycobacterium TB as the
cause of tuberculosis in 1892, but the strains he studied were
not preserved, and it is unknown what happened to them.
Koch discovered Mycobacterium tuberculosis as the cause
of tuberculosis in 1892, but the strains he studied were not
preserved, so it's unclear how closely H37Rv is related to
those strains. H37Rv has remained the most widely used
tuberculosis strain in laboratories, and its whole genome
was revealed for the first time in 1998. It does not, however,
have some characteristics found in recent clinical isolates,
such as the capacity to produce caseous necrosis in rabbits.
It does not, however, have some characteristics found in
recent clinical isolates, such as the capacity to produce
caseous necrosis in rabbits. Strains produced from H37Rv
in different labs have also been found to evolve over time,
with one study of six strains revealing between five and 10
polymorphisms per strain. These comprised IS6110
transposable element insertions and deletions that changed
the spoligotype of the strain. The authors of the research
cautioned against using all H37Rv strains as a reference
since there might be significant differences depending on
the laboratory where it is housed.
ROLE OF PP AND PPE PROTEINS IN HOST
PATHOGEN INTERACTION
Table 1 summarises the function and location of PE/PPE
proteins based on the distinct stages of contact with the host,
and Figure 1A highlights the fascinating roles: I surviving
intracellular stress, phagocytosis, and phagolysosome
maturation; (ii) cell fate determination
ROLE OF PPE PROEINS IN INTERACTION WITH
HOST CELLS AND IMMUNE REGULATION
PE/PPE proteins can interact directly with their host targets
after being exposed to the surface or secreted into the
extracellular environment. Some proteins are said to bind
with receptors on macrophages' surfaces, such as TLR2/4,
triggering downstream signalling cascades. TLR4 interacts
with PE9-PE10 and PE PGRS5 to trigger downstream
signalling and alter cytokine production. PPE26, PPE32,
PPE57, PPE65, PE PGRS33, and PE PGRS11 are only a
few of the PE/PPE proteins that can interact with TLR2.
PPE18 has been shown to promote IL-10 release, which
may generate a Th2 response when it interacts with TLR2,
as well as limit the generation of NF-kB/rel-mediated pro-
inflammatory cytokines by upregulating suppressor of
cytokine signalling 3 protein (SOCS3). PE PGRS17 was
also discovered to mature DCs via TLR2 and trigger host
cell death and cytokine production via Erk kinase, resulting
in improved intracellular survival. PE/PPE protein binding
to cell surface receptors triggers downstream signalling
pathways such as NF-kB and MAPK (p38, JNK, and ERK),
which influence cytokine production and result in a pro-
inflammatory or anti-inflammatory response. th was
overexpressed by PPE27. The PPE27 overexpressed strain
had a stronger capacity to produce NO and reduce IL-6
production, which was blocked by inhibitors of NF-kB, p38,
and ERK. PPE39, a PE/PPE protein identified in the
hypervirulent strain Beijing/K, was found to mature DCs
and stimulate the Th1 immune response via NFkB and
MAPK, a TLR4 agonist. PE13, PE27, PPE26, PPE32,
PPE44, PPE57, PE PGRS11, and PE PGRS17 are among
the proteins that modulate the cytokine profile via NF-kB
and MAPK signalling. It's also been proposed that PE/PPE
proteins have an influence on mycobacterial invasion and
macrophage phagocytosis. The invasion efficiency of the
PPE38- mutant of Mycobacterium marinum was much
greater, but the phagocytosis ratio of PPE29 mutants was
lowered as predicted. Another need for bacterial invasion is
adhesion to the cell surface. PE11 knockdown strains have
been shown to increase the synthesis of fibronectin
attachment protein, which aids in the attachment to the host
extracellular matrix. PE PGRS60 has the ability to bind to
fibronectin, resulting in increased adhesion and invasion.
ROLE OF PP/PPE PROTEINS IN INTRACELLULAR
SURVIVAL
Pathogens adapt to the intracellular environment, such as
low pH, reactive oxygen, and nitrogen species, as they enter
macrophages, and so create their own niche. PPE60 and
PE13 can also boost intracellular survival by increasing cell
resilience to low pH, surface stressors, and antibiotic
exposure. PPE11 also maintains a high bacterial load in
mouse tissue, exacerbating organ disease, and increases
early bacterial survival rate under circumstances
comparable to the internal macrophage environment, such
as the presence of lysozymes, acidity, and active nitrogen
intermediates (RNI).
Mtb lives in macrophages by blocking phagosomal
acidification and phagosome-lysosome fusion after it has
acclimated to the harsh circumstances. The knockout strains
PE PGRS30 and PE PGRS47 lost their capacity to suppress
phagosome fusion. Similarly, overexpression of PE
PGRS62 prevents phagosome maturation. PPE25
transcription is increased during phagocytosis, and the

PPE25 mutant strain loses its capacity to proliferate within
macrophages and prevents phagosome-lysosome union.
Furthermore, a PPE60-overexpressing strain has been
discovered to boost intracellular survival and change cell
destiny to pyroptosis, a newly described form of
programmed cell death that is linked to intracellular growth
limitation, heightened host immunological response, and
IL-1b and IL-18 maturation.
•Pathogenic, slow-growing Mycobacterium
tuberculosis and other M. tuberculosis complex
(MTBC) species include the pe/ppe genes. These
genes are important in the interactions between the
host and the pathogen. Despite the fact that the
function of most PE/PPE family proteins is
unknown, mounting evidence shows that they are
involved in M. tuberculosis infection.
•PPE22 At 30 days, mass spectrometry identified it
in M. tuberculosis H37Rv-infected guinea pig lungs,
but not at 90 days. M. tuberculosis H37Rv whole cell
lysates, but not the culture filtrate or membrane
proteinfraction, were identified by mass
spectrometry.
•Rv1403c Intermediary respiration and metabolism
Mutant In vitro development of H37Rv in a MtbYM
rich media requires this non-essential gene. H37Rv
tuberculosis Rv1403c-Rv1404-Rv1405cm utant
develops at a slower rate than wild-type
1. PPE 22
Protein Sequence
MDFGALPPEVNSGRMYCGPGSAPMVAAASAW
NGLAAELSVAAVGYERVITTLQTEEWLGPASTL
MVEAV
APYVAWMRATAIQAEQAASQARAAAAAYETA
FAAIVPPPLIANRARLTSLVTHNVFGQNTASIAA
TEAQYA EMWAQDAMA
MYGYAGSSATATKVTPFAPPPNTTSPSAAATQL
SAVAKAAGTSAGAAQSAIAELIAHLPN
TLLGLTSPLSSALTAAATPGWLEWFINWYLPISQ
LFYNTVGLPYFAIGIGNSLITSWRALGWIGPEAA
EAAAA APAAVGA
AVGGTGPVSAGLGNAATIGKLSLPPNWAGASPS
LAPTVGSASAPLVSDIVEQPEAGAAGNLLGGMP
LAGS
GTGTGGAGPR YGFRVTVMSR PPFAG
2. Rv1403c
Protein sequence:
MTVYTPTSERQAPATTHRQMWALGDYAAIAEE
LLAPLGPILVSTSGIRRGDRVLDVAAGSGNVSIP
AAMAG
AHVTASDLTPELLRRAQARAAAAGLELGWREA
NAEALPFSAGEFDAVLSTIGVMFAPRHQRTADE
LARVC
RRGGKISTLNWTPEGFYGKLLSTIRPYRPTLPAG
APHEVWWGSEDYVSGLFRDHVSDIRTRRGSLT
VDRFG
CPDECRDYFKNFYGPAINAYRSIADSPECVATLD
AEITELCREYLCDGVMQWEYLIFTARKC
III. METHODOLOGY
1. Selection of M.tb strain, Antigens and
retrieval of protein sequences
2. To check the sub localization of protein
3. To design Primer for Gene PPE 22 and
Rv1403c
4. PCR Reaction of Genes
5. PCR Product Separation by Gel
Electrophoresis
6. Extraction OF Amplified Products
7. Preparation of Competent cells
8. Insert of Gene of Interest in competent
cells
9. Blue and white selection of
Transformants and non-transformants
Selection of M.tb strain, Antigens and retrieval of
protein sequences.
M.tb strain is selected on the basis of previous work
related to selection of vaccine candidate that is lab
strain H37Rv .Antigen can be selected on the basis of
which protein show up regulation. Antigen choosed
from the H37Rv and its basic information like Gene
Sequence , protein Sequence, molecular weight ,
promoter etc. were find through mycobrowser
application.(..mycobrowser.html).
To check the sub localization of protein
To determine whether or not proteins are present
within the cell. psortb (..psort b.html) is a programme
that evaluates the amino acid composition of a query
protein sequence and compares it to proteins of known
localization, the presence of a signal peptide,
transmembrane alpha-helices, and motifs related to
specific localizations.
To design Primer for Gene PPE 22 and Rv1403c

Primer is required for the amplification of Rv2628 in PCR.
Primer is designed on the basis of some properties like
length (17-30 bp), GC content (more than 50%), melting
temperature difference between forward and reverse primer
is less than 5℃, hair loop formation, self dimmers should
be checked. Different software are used to make primers
like Primer 3, oligoanalyzer(IDT)
(..oligoanalyzerIDT.html) to check whether designed
primers at different parameters. After primer designing add
the restriction endonuclease which is non cutter for Rv2628
(..NEB.html) in the primers for further cloning process.
Designing primers PPE 22
• Forward Primer sequence (5’ – 3’)
ATG GAT TTT GGG GCG TTG CCA CCG
Length: 24
GC content: 58.3%
Melting temperature: 64.7 ºC
Melting temperature of hairpin loop structure:
47.9ºC
• Reverse Primer sequence (5’ – 3’)
TTA TCC GGC AAA CGG CGG CCG
Length: 21
GC content: 66.7%
Melting temperature of hairpin loop structure:
57.1 ºC
Primer Designing Rv1403c
Forward primer: 5’
ATGACTGTCTACACACCCACC 3’
GC content: 52.4 %
Hairpin: Tm = 20.4◦C
Self-dimer: no significant self-dimers
Reverse primer: 5’
TCAACACTTCCGGGCGGT 3’
GC content: 61.1 %
Hairpin: Tm = 31.6◦C
Self-dimer: Only 1 significant self-dimers
PCR Reaction of Genes
The polymerase chain reaction (PCR) is a
technique for producing vast amounts of DNA.
• A DNA target (100-35,000 bp in length).
• Two complementary primers (synthetic
oligonucleotides of 17-30 nucleotides length)
surrounding the target DNA.
• dATP, dCTP, dGTP, and dTTP are the four
deoxyribonucleotides.
• A DNA polymerase that can endure
temperatures of up to 95 degrees Celsius
• Denaturation: When the temperature is raised
to around 95°C for 1 minute, the DNA
denatures and the two strands split.
• Renaturation or annealing: The primers base
pair with complementary areas flanking target
DNA strands when the temperature of the
mixture is gently lowered to around 55°C.
Synthesis
The 3'-hydroxyl end of each primer is where
DNA synthesis begins.
- The primers are lengthened by connecting
complimentary bases on DNA strands.
- The synthetic process is similar to leading
strand DNA replication.
- DNA polymerases need that the temperature
be kept at a certain level.
-Taq DNA polymerase has an optimal
temperature of 75°C, while E.coli DNA
polymerase has an optimum temperature of
37°C.
-Raise the temperature to to 95°C to cease the
process. -Each PCR cycle lasts roughly 3-5
minutes.
Reaction Mixture Preparation
NFW- 26.7 µl
Phusion Buffer- 10µl
10mM DNTP- 1µl
10µM FP – 2.5µl
10µM RP- 1.5µl
DMSO- 1.5µl
Phusion Polymerase- 0.8µl
Template – 5µl
PCR Product Separation by Gel Electrophoresis

International Journal of Medical, Pharmacy and Drug Research (IJMPD)
[Vol-6, Issue-2, Mar-Apr, 2022]
ISSN: 2456-8015
https://dx.doi.org/10.22161/ijmpd.6.2.6
Peer-Reviewed Journal
Extraction OF Amplified Products
• Using a clean scalpel or razor blade, remove the
gel slice containing the DNA fragment. To reduce
the gel volume, cut as near to the DNA as feasible.
Place the gel slice in an eppendrof and that has
been pre-weighed, and then weigh it.
• To the gel slice, add a 1:1 volume of binding buffer
(volume: weight).
• Incubate the gel mixture for 10 minutes at 50-
60°C, or until the gel slice is fully dissolved. To
speed up the melting process, invert the tube every
few minutes. Check to see if the gel has totally
dissolved. Before placing the gel mixture onto the
column, vortex it briefly.
• Fill the Gene Jet Purification column with up to
800 l of the solubilized gel solution. For 1 minute,
centrifuge at 10,000 rpm. Remove the flow-
through and re-insert the column into the collecting
tube.
• Quantify the sample with Nanodrop before moving
on to the next step.
Preparation of Competent cells
• Isolate E.coli DH5 colonies on LBM Plates
(without ampicillin) and incubate at 37°C
overnight (16-20hours).
• Collect cells from a single colony with a sterile
inolculating loop and inoculate 50 ml sterile 1X
LBM Broth to grow at 37°C overnight in an
incubator shaker. To equilibrate the temperature of
the medium, place 2 flasks of 250 ml of 1X LBM
in the incubator.
• Fill each 250 mL flask with 25 mL of the overnight
culture. To equilibrate the temperature of the
medium, place another flask of 150 ml 1X LBM in
the incubator. The cultures should be grown to an
OD650 of 0.2. In each flask, add 75 mL of
equilibrated 1X LBM and incubate for 30 minutes.
Sample was prepared by adding 80µl of autoclaved distilled water and 20
µl of loading solution ( glycerol : TAE Buffer (1:4) )
A 1% (w/v) Agarose gel was prepared by dissolving Agarose powder in
1X Tris-Acetate -EDTA buffer (TAE).
20 µl of each sample was loaded and run electrophoresis at 80V for 1-2
hour
Gel is visualized under visible light

• Pellet the cells in a cold autoclaved eppendroff and
centrifuge for 10 minutes at 5000 rpm.
Resuscitations can then be done in the same bottle.
• Finally, place the tubes holding the cells on ice in
the cold chamber.
• Decant the supernatant and resuspend the cells in
100mM Mgcl2 at freezing temperature for 5
minutes. Transfer the cells to sterile centrifuge
tubes that have been pre-chilled, then centrifuge at
4000rpm for 10 minutes at 4°C.
• Drain the supernatant and resuspend the cells in ice
cold 100mM Cacl2, then incubate for 20 minutes
on ice. Pellet the cells by centrifuging them for 10
minutes at 4°C at 4000rpm.
• Drain the supernatant and resuspend the cells in a
mixture of 85 percent v/v Cacl2 and 15% v/v
glycerol. And I kept it in an eppendorf freezer at -
80°C for later use.
Insert of Gene of Interest in competent cells
• Put the competent cells on ice to thaw.
• In a 1.5 ml microcentrifuge tube, chill
approximately 5ng (2l) of the ligation mixture.
• Toss in 50 litres of competent cells with the DNA.
Pipette up and down 4-5 times to gently mix the
cells; do not vortex.
• Freeze the mixture for 30 minutes. Do not
combine.
• Heat shock for 30 seconds at 42°C. Do not
combine.
• Fill the tube with 950 l of room temperature
medium.
• Preheat the oven to 37°C and bake the tube for 60
minutes. Rotate or shake vigorously.
• Preheat the selection plates to 37°C.
• Cover the plates with 50-100 l of the cells and
ligation mixture.
• Incubate at 37°C overnight.
Blue and white selection of Transformants and non-
transformants
• Prepare and autoclve LB Agar. Reduce the
temperature of the autoclaved growth medium agar
to 50°C.
• Make an IPTG solution of 100mM and an X-Gal
solution of 20mg/ml.
• To attain a final concentration of 1mM, add 10l X-
Gal solution per 1 ml medium and 10l per ml
media.
• Add ampicillin as a screening antibiotic.
• Pour the mixture onto the dishes and let aside to
cool to room temperature.
• As needed, disperse the changed competent cells.
IV. FUTURE APPLICATIONS OF PE/PPE
FAMILY PROTEINS IN TB VACCINE
DESIGN AND DIAGNOSTIC TOOL
DEVELOPMENT
Although serological antibody tests are commonly used,
there is no gold standard for TB diagnosis. PE35, an RD1-
encoded antigen, can tell the difference between pulmonary
and extrapulmonary tuberculosis patients and healthy BCG-
vaccinated people. Another noteworthy example is PPE17,
whose N-terminal elicits a significant immunogenic
response and has more potential to be a sero-diagnostic
marker than full-length PPE17, which can screen latently
infected individuals. PPE2 might be used as a
serodiagnostic marker to identify extrapulmonary and
smear-negative pulmonary patients. The examination of
IFN-g T cell responses elicited after infection indicated the
highly immunogenic features of PE/PPE proteins. PE18,
PE19, PPE25, PPE26, and PPE27, which are CD4+-specific
epitope-rich PE/PPE proteins, are strong inducers of cell-
mediated immune responses. During human and bovine
infection, Vordermeier et al. looked at cellular immune
responses to a panel of 36 PE/PPE proteins and found that
several of them were key targets of the cellular immune
response to TB. PPE68 epitopes that are HLAA*0201-
restricted also induce a strong cellular response. In addition,
during intradermal testing, the PE5 protein and EsxI have
been shown to be a diagnostic antigen for bovine TB. In the
IFN-g releasing test for identifying active TB, a
combination of PPE57 can improve the sensitivity of
ESAT-6 or CFP-10. PE/PPE proteins may be superior
diagnostic and vaccine options for intracellular survival of
bacteria due to the more cellular immune response. PE/PPE
proteins are also polymorphic among clinical isolates and
can be resistant to degradation, which limits MHC
processing. Surprisingly, researchers discovered that the
PPE18 protein, which increased IL-10 production while
inhibiting the inflammatory response, may be investigated
as a treatment for sepsis caused by excessive inflammatory
reactions. The utilisation of immunodominant epitopes of
PE/PPE proteins or thorough characterization of candidates
may make vaccine production easier.

V. DISCUSSION
PE/PPE family has been regarded as unique to
mycobacteria, especially pathogenic species, since its
discovery over 20 years ago. PE/PPE protein expression is
now well-established as being connected to ESX gene
clusters, according to several studies. Our understanding of
the biological role of individual PE/PPE proteins has greatly
improved as our understanding of the ESX system has
improved. Furthermore, structural biology research has
begun to unravel and explain the functions of protein
complexes involved in PEPPE and ESX secretion. PE/PPE
proteins' function and structure, on the other hand, are
significantly less well studied than other mycobacterial
proteins. The structure of PE/PPE proteins and their
interactions with ESX systems will be crucial in gaining a
better understanding of how the PE/PPE protein family, in
conjunction with the ESX secretion system, contributes to
Mtb pathogenicity. This is critical for learning more about
mycobacteria's virulence methods, and it might lead to the
discovery of new antimycobacterial targets.
Another characteristic of PE/PPE proteins is that they are
frequently discovered as co-operonic pairs comprising
primarily one PE- and one PPEcoding gene, whose products
interact and are thought to assemble as heterodimers. As in
the cases of PPE41 and PE25, PE35 and PPE68, and PE19
and PPE51, such interactions have been predicted using
bioinformatic methods and validated by experimental data.
PPE51 deletion caused Mtb cells unable to proliferate in
propionamide, glucose, or glycerol, according to Korycka-
Machaa et al. Mtb also requires several PE/PPE proteins
during Mg2+ and PO32 restriction, such as PE20/PPE31
and PE32/PPE65. PPE36/PPE62 and PPE37 are required
for Mtb growth and heme iron accumulation. In addition,
mutant PPE51 and PE19 bacteria showed resistance to
3bMP1, a tuberculosis-fighting chemical. These findings
show that at least some PE/PPE proteins operate as solute-
selective pores, enabling exogenous chemicals or nutrients
necessary for proliferation to pass through. Thus,
concentrating on pe/ppe family genetic alterations, which
are frequently overlooked when evaluating next-generation
sequencing data of clinically drug-resistant populations,
may aid in the discovery of antituberculosis drug resistance
mechanisms. In conclusion, we anticipate that research into
the PE/PPE family will continue to be quite busy, with many
fascinating aspects still to be found.
VI. CONCLUSION
Mycobacteria have a strong protective outer coating that
protects them from the elements both outside and within the
host. However, the bacteria must produce proteins over this
outer barrier in order to cause illness. Mycobacteria have
type VII secretion systems that help them do this. The ESX-
5 secretion system, for example, is found only in the group
of slow-growing mycobacteria, which includes the majority
of harmful species. The ESX-5 system is required for
mycobacteria development, according to this research. The
ESX-5 system is required for mycobacteria development,
according to this research. The ESX-5 system was no longer
required for development when we created a 'leaky' outer
membrane by interfering with the outer membrane's
formation or adding an outer membrane porin. We also
discovered that ESX-5 promotes fatty acid absorption,
implying that ESX-5 substrates can build particular
transport networks or holes in the outer membrane essential
for nutrient uptake. Understanding the involvement of ESX-
5 in outer membrane permeability sheds light on a key
distinction between fast- and slow-growing mycobacteria.
Because most pathogenic mycobacteria are slow-growing,
we can better grasp what mycobacteria need to cause
disease.
REFERENCES
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